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. 2018 Mar 1;110(3):304-315.
doi: 10.1093/jnci/djx166.

Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis (V体育2025版)

Yibing Yang  1 , Xinya Gao  1 , Maolei Zhang  1 , Sheng Yan  1 , Chengjun Sun  1 , Feizhe Xiao  1 , Nunu Huang  1 , Xuesong Yang  1 , Kun Zhao  1 , Huangkai Zhou  1 , Suyun Huang  1 , Bo Xie  1 , Nu Zhang  1
Affiliations

Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis

Yibing Yang et al. J Natl Cancer Inst. .

Erratum in

  • Corrigendum. (VSports在线直播)
    [No authors listed] [No authors listed] J Natl Cancer Inst. 2018 Oct 1;110(10):1147. doi: 10.1093/jnci/djy118. J Natl Cancer Inst. 2018. PMID: 29986068 Free PMC article. No abstract available.

Abstract

Background: Circular RNAs (circRNAs) are RNA transcripts that are widespread in the eukaryotic genome. Recent evidence indicates that circRNAs play important roles in tissue development, gene regulation, and carcinogenesis VSports手机版. However, whether circRNAs encode functional proteins remains elusive, although translation of several circRNAs was recently reported. .

Methods: CircRNA deep sequencing was performed by using 10 pathologically diagnosed glioblastoma samples and their paired adjacent normal brain tissues V体育安卓版. Northern blotting, Sanger sequencing, antibody, and liquid chromatograph Tandem Mass Spectrometer were used to confirm the existence of circ-FBXW7 and its encoded protein in in two cell lines. Lentivirus-transfected stable U251 and U373 cells were used to assess the biological functions of the novel protein invitro and invivo (five mice per group). Clinical implications of circ-FBXW7 were assessed in 38 pathologically diagnosed glioblastoma samples and their paired periphery normal brain tissues by using quantitative polymerase chain reaction (two-sided log-rank test). .

Results: Circ-FBXW7 is abundantly expressed in the normal human brain (reads per kilobase per million mapped reads [RPKM] = 9. 31). The spanning junction open reading frame in circ-FBXW7 driven by internal ribosome entry site encodes a novel 21-kDa protein, which we termed FBXW7-185aa. Upregulation of FBXW7-185aa in cancer cells inhibited proliferation and cell cycle acceleration, while knockdown of FBXW7-185aa promoted malignant phenotypes invitro and invivo. FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28-induced c-Myc stabilization. Moreover, circ-FBXW7 and FBXW7-185aa levels were reduced in glioblastoma clinical samples compared with their paired tumor-adjacent tissues (P < V体育ios版. 001). Circ-FBXW7 expression positively associated with glioblastoma patient overall survival (P = . 03). .

Conclusions: Endogenous circRNA encodes a functional protein in human cells, and circ-FBXW7 and FBXW7-185aa have potential prognostic implications in brain cancer VSports最新版本. .

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Figures

Figure 1.
Figure 1.
Profiling of circular RNAs in human glioblastoma and adjacent normal tissues. A) RNA-seq read abundance distribution of identified circular RNAs (circRNAs). x-axis: the back-spliced read numbers of circRNAs detected in RNA-seq. y-axis: the abundance of circRNAs classified by different read numbers. The majority of called circRNAs in the study were supported by more than 10 reads. B) Venn plot showing the number of circRNAs derived from different genomic regions. C) Length distribution of the identified circRNAs. x-axis: the length of circRNAs detected in this study. y-axis: the abundance of circRNAs classified by different lengths. D) The distribution of identified circRNAs in chromosomes. The yellow and cyan bars represent the location of detected circRNAs within different chromosomes in normal and tumor samples, respectively. E) Numbers of identified circRNAs in different chromosomes. The yellow and cyan bars represent the numbers of circRNAs within different chromosomes detected in normal and tumor samples, respectively. F) Heat map of all differentially expressed circRNAs between normal and tumor tissues. PR-MIX = tumor peripheral RNA mix; TR-MIX = tumor RNA mix.
Figure 2.
Figure 2.
Characterization of circ-FBXW7 as a circular RNA in vitro and in vivo. A) In the volcano plot, the green, red, and black points represent downregulated, upregulated, and no statistically significant difference circular RNAs (circRNAs) in the TR-MIX group compared with the PR-MIX group, respectively. x-axis: log2 ratio of circRNA expression levels between normal and tumor tissues. y-axis: the false discovery rate value (-log10 transformed) of circRNAs. The blue dot indicates novel_circ_022705 (circ-FBXW7). B) Upper panel, illustration of the annotated genomic region of FBXW7, the putative different RNA splicing forms, and the validation strategy for circular exon 3 and 4 (circ-FBXW7). Convergent (blue) and divergent (red) primers were designed to amplify the linear or back-splicing products. Lower panel: total RNA from 293T cells with or without RNase-R treatment were subjected to polymerase chain reaction (PCR). C) Expression levels of circ-FBXW7 in 100 randomly selected glioma samples and 100 normal brain tissues were detected by the junction primers (P < .001). Values are the average ± SD of three independent experiments. The Student’s two-tailed unpaired t test was used to determine statistical significance between the normal and glioma groups. D) Sanger sequencing following PCR conducted using the indicated divergent flanking primers confirmed the “head-to-tail” splicing of circ-FBXW7 in 293T cells. E) Upper panel: illustration of the synthetic circRNA expression plasmid. I: exons 3 and 4 of the FBXW7 gene were cloned between splicing acceptor (SA) and splicing donor (SD) sequences with upstream flanking repeat sequences (black arrows) (sequences showed in Supplementary Table 1, available online). II: exon 3 and 4 sequences of the FBXW7 gene were cloned between SA and SD with both sides of flanking repeat sequences. III: compared with vector II, the putative internal ribosomal entrance site sequences were mutated (as shown in Supplementary Figure 2, available online). Lower panel: empty vector and above-mentioned vectors were transfected into 293T cells. After 24 hours of transfection, total RNA was treated with RNase-R and subjected to Northern blotting using circ-FBXW7 junction probes. F) Upper panel: fluorescence in situ hybridization with junction-specific probes indicates the cellular localization of circ-FBXW7. Scale bars = 5 μM. Lower panel: circ-FBXW7 was detected in different cell fractions. Nuclear and cytoplasmic RNA was extracted, and junction primers were used for circ-FBXW7 detection. U6 was used as internal control of nuclear RNA, and GAPDH was used as internal control for cytoplasmic RNA. Values are the average ± SD of three independent experiments. The Student’s two-tailed unpaired t test was used to determine the statistical significance between nuclear and cytoplasm circ-FBXW7 expression. EV = transection of empty vector; M = nucleotide marker; NC = negative control.
Figure 3.
Figure 3.
Evaluation of the coding ability of circ-FBXW7. A) Upper panel: the putative open reading frame (ORF) in circ-FBXW7. Note that the circ-FBXW7 junction is inside the ORF. Lower panel: the sequences of the putative ORF are shown in green, internal ribosomal entrance site (IRES) sequences are shown in purple, and specific amino acid sequences of FBXW7-185aa are shown in yellow. B) The putative IRES activity in circ-FBXW7 was tested. Upper panel: IRES sequences in circ-FBXW7 or its different truncations were cloned between Rluc and Luc reporter genes with independent start and stop codons. Lower panel: the relative luciferase activity of Luc/Rluc in the above vectors was tested. Values are the average ± SD of three independent experiments. C) Upper left: vector set for detecting circ-FBXW7 encoded protein. Endo-circ-FBXW7: illustration showing how endogenous circ-FBXW7 formed; the circular junction is inside the ORF and formed the unique sequences shown in yellow. Circ-FBXW7-FLAG: exon 3 and exon 4 of FBXW7 were cloned between splicing acceptor, splicing donor, and side flanking repeat sequences (black arrows); the circular junction was moved to the stop codon of the ORF. FLAG tag was divided to both sides (light and dark blue). Circularization of this vector could form the same circular RNA as endogenous circ-FBXW7, except with a FLAG tag added behind the ORF. Linear-FL-FBXW7-AG: circ-FBXW7-FLAG vector lacking the downstream flanking repeat sequence. FBXW7-185aa-FLAG: the 185 aa ORF with FLAG tag was directly cloned inside a linear expression vector. Middle: the putative FBXW7-185aa amino acid sequences and the antigen used to produce the FBXW7-185aa antibody. The red amino acids were distinctly formed by the circ-FBXW7 junction. Lower: FLAG tag antibody and FBXW7-185aa antibody were used to detect FBXW-185aa expression in 293T cells transfected with the above vectors. D) Total proteins from circ-FBXW7 or control plasmid-transfected 293T cells were separated via SDS-PAGE (left). FBXW7-185aa overexpression was confirmed by immunoblotting (upper right). The differential gel bands between 17 kDa and 26 kDa were extracted and subjected to liquid chromatograph Tandem Mass Spectrometer. Among the liquid chromatograph Tandem Mass Spectrometer results, two FBXW7-185aa junction-specific peptides were identified (lower right).
Figure 4.
Figure 4.
Tumor-suppressive functions of FBXW7-185aa in glioma cell lines. A) circ-FBXW7 expression was detected by using junction primers in U251 and U373 cells stably expressing circ-FBXW7 or circ-FBXW7 internal ribosomal entrance site (IRES) mut plasmids. B) FBXW7-185aa expression was detected in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. C) Cell cycle was determined in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. D) The growth curve was determined by MTT assay in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. E) Left panel: circ-FBXW7 or circ-FBXW7-IRES mut overexpressing U251 and U373 cells were treated with 10 µM EdU for two hours, then detected with Andy Fluor 594 azide (red); cells were counterstained with DAPI (blue). Scale bar = 50 μM. Right panel: quantification of left panel. The percentage of EdU-incorporated cells was counted in 200 cells. F) Two spanning junction shRNAs were designed for circ-FBXW7 and stably expressed in Hs683 and SW1783 anaplastic astrocytoma cell lines. The RNAi effect was determined by quantitative polymerase chain reaction using junction primers for circ-FBXW7. FBXW7-185aa expression was detected in circ-FBXW7 shRNA stably expressing Hs683 and SW1783 cells and their control cells. G) The cell cycle was determined in sh-circ-FBXW7 stably expressing Hs683 and SW1783 cells and their control cells. H) The growth curve was determined by MTT assay of sh-circ-FBXW7 stably expressing Hs683 and SW1783 cells and their control cells. I) Left panel: circ-FBXW7 stable knockdown Hs683 and SW1783 cells and their respective control cells were treated with 10 µM EdU for two hours, then detected with Andy Fluor 594 azide (red); cells were counterstained with DAPI (blue). Scale bar = 50 μM. Right panel: quantification of left panel. The percentage of EdU-incorporated cells was counted in 200 cells. In each panel of this figure, values are the average ± SD of three independent experiments. The Student’s two-tailed unpaired t test was used to determine statistical significance in/between indicated groups.
Figure 4.
Figure 4.
Tumor-suppressive functions of FBXW7-185aa in glioma cell lines. A) circ-FBXW7 expression was detected by using junction primers in U251 and U373 cells stably expressing circ-FBXW7 or circ-FBXW7 internal ribosomal entrance site (IRES) mut plasmids. B) FBXW7-185aa expression was detected in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. C) Cell cycle was determined in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. D) The growth curve was determined by MTT assay in circ-FBXW7 or circ-FBXW7 IRES mut plasmid stably overexpressing U251 and U373 cells. E) Left panel: circ-FBXW7 or circ-FBXW7-IRES mut overexpressing U251 and U373 cells were treated with 10 µM EdU for two hours, then detected with Andy Fluor 594 azide (red); cells were counterstained with DAPI (blue). Scale bar = 50 μM. Right panel: quantification of left panel. The percentage of EdU-incorporated cells was counted in 200 cells. F) Two spanning junction shRNAs were designed for circ-FBXW7 and stably expressed in Hs683 and SW1783 anaplastic astrocytoma cell lines. The RNAi effect was determined by quantitative polymerase chain reaction using junction primers for circ-FBXW7. FBXW7-185aa expression was detected in circ-FBXW7 shRNA stably expressing Hs683 and SW1783 cells and their control cells. G) The cell cycle was determined in sh-circ-FBXW7 stably expressing Hs683 and SW1783 cells and their control cells. H) The growth curve was determined by MTT assay of sh-circ-FBXW7 stably expressing Hs683 and SW1783 cells and their control cells. I) Left panel: circ-FBXW7 stable knockdown Hs683 and SW1783 cells and their respective control cells were treated with 10 µM EdU for two hours, then detected with Andy Fluor 594 azide (red); cells were counterstained with DAPI (blue). Scale bar = 50 μM. Right panel: quantification of left panel. The percentage of EdU-incorporated cells was counted in 200 cells. In each panel of this figure, values are the average ± SD of three independent experiments. The Student’s two-tailed unpaired t test was used to determine statistical significance in/between indicated groups.
Figure 5.
Figure 5.
The molecular mechanism of FBXW7-185aa in suppressing glioma tumorigenesis. A) FBXW7-185aa and c-Myc expression were detected by immunoblotting of circ-FBXW7 or circ-FBXW7 internal ribosomal entrance site mut overexpressed U251 and U373 cells. B) Upper panel: FBXW-185aa or control plasmid-transfected U251 cells were treated with cycloheximide. Total lysates at the indicated time points were collected, and indicated proteins were determined by immunoblotting. Lower panel: semiquantification of c-Myc protein levels in the above immunoblotting. Values are the average ± SD of three independent experiments. C) GST-tagged FBXW7-185aa and 6XHis-tagged USP28 were expressed in Escherichia coli. Purified His-tagged-USP28 and GST-tagged-FBXW7-185aa were subjected to in vitro GST or His pull-down assays. The pull-down lysates were subjected to immunoblotting. D) In vivo interaction of USP28 and FBXW7-185aa was detected by immunoprecipitation in SW1783 cells. E) HA-tagged-FBXW7α and His-tagged-USP28 were cotransfected with increasing doses of FLAG-tagged-FBXW7-185aa. Immunoprecipitation was performed using anti-HA or anti-His antibody, followed by immunoblotting using the indicated antibodies. F) FLAG-tagged-FBXW7-185aa, His-tagged-Ubiquitin, USP28 and HA-tagged-Myc were transfected into 293T cells at the indicated combinations. Immunoprecipitation was performed using anti-HA antibody, and the ubiquitination level of c-Myc was determined by immunoblotting. G) FBXW7-185aa-overexpressing Hs683 or SW1783 cells were stably transfected with sh-FBXW7α or USP28 plasmids. After 72 hours, c-Myc, FBXW7α, FBXW7-185aa, and USP28 levels were detected by immunoblotting. All statistical tests were two-sided. CHX = cycloheximide.
Figure 6.
Figure 6.
Clinical implications of circ-FBXW7 and FBXW-185aa. A) Six randomly selected glioblastoma patient samples and their paired adjacent normal brain tissues were collected. Circ-FBXW7 and FBXW7-185aa were detected by quantitative polymerase chain reaction (q-PCR) and immunoblotting, respectively. Values are the average ± SD of three independent experiments. The Student’s two-tailed unpaired t test was used to determine statistical significance between indicated groups. B) Upper panel: circ-FBXW7 levels in cancerous and adjacent normal tissues were detected in a cohort of 38 glioblastoma patients by q-PCR. Lower panel: patients in the cohort were divided into two groups according to relative circ-FBXW7 expression. The overall survival time of each group was calculated. The P value between the circ-FBXW7-high and -low groups is .03 (log-rank test) or .04 (Gehan-Breslow-Wilcoxon test). C) Circ-FBXW7 or circ-FBXW7-internal ribosomal entrance site mut plasmid stably overexpressing U251 and U373 cells were intracranially injected into nude mice; 5 × 105 cells were used per mouse, and each group contained five mice. Mice were killed, and the brain tissues were collected if clinical symptoms, such as emaciation, disorientation, or hemiplegia, were shown. At day 100, all mice were killed, and hematoxylin and eosin (HE) staining was performed on all collected brain tissues. Representative HE staining of brain sections in each experimental group is shown. Tumors are indicated by white arrows. D) The survival time in each experimental group was calculated. The survival time of mice injected with circ-FBXW7-overexpressed glioblastoma multiforme cells was longer than the mice injected with corresponding control cells (n = 5 mice per group, P < .001, Log rank test). All statistical tests were two-sided.
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"V体育2025版" References

    1. Salzman J, Gawad C, Wang PL et al. , Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One. 2012;72:e30733. - PMC - PubMed
    1. Jeck WR, Sorrentino JA, Wang K et al. , Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2013;192:141–157. - PMC - PubMed
    1. Jeck WR, Sharpless NE.. Detecting and characterizing circular RNAs. Nat Biotechnol. 2014;325:453–461. - PMC - PubMed
    1. Nigro JM, Cho KR, Fearon ER et al. , Scrambled exons. Cell. 1991;643:607–613. - PubMed (V体育2025版)
    1. Capel B, Swain A, Nicolis S et al. , Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993;735:1019–1030. - PubMed

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